CN112125827A - A kind of synthetic method for carbonyl-containing steroid compound derivatization reagent - Google Patents

Abstract

The invention relates to a synthesis method for a carbonyl-containing steroid compound derivatization reagent, and belongs to the technical field of chemical synthesis. The synthesis method is to first utilize 1,3-dibromopropane and N-hydroxyphthalimide to react to synthesize 2-(3-bromopropoxy)isoindoline-1,3-dione; and then use 2 -(3-Bromopropoxy)isoindoline-1,3-dione to prepare 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N, N-trimethylpropylammonium bromide; finally to 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropyl bromide ammonium chloride for hydrazinolysis. The synthesis method of the invention has the advantages of low cost, simple steps and high yield, and can derivatize the carbonyl-containing steroid compound, introduce a permanent charged group-N ⁺ Me ₃ into it, and improve the ESI-MS/MS/MS ratio of the carbonyl-containing steroid compound. Detection sensitivity in MS.

Description

A kind of synthetic method for carbonyl-containing steroid compound derivatization reagent

technical field

The invention belongs to the technical field of chemical synthesis, and more particularly relates to a method for synthesizing a derivatizing reagent for carbonyl-containing steroid compounds.

Background technique

Carbonyl-containing steroids are a class of non-polar compounds that are secreted by the endocrine system and regulate metabolic processes, and have important physiological functions. Due to the low concentration of carbonyl-containing steroids in biological samples, poor ionization efficiency and many isomers, their analysis faces great challenges. At present, the derivatization strategy is often used to analyze such compounds, and the introduction of ionizable groups in the molecule can significantly improve its sensitivity. Commonly used derivatizing reagents include dansyl chloride (DNS-Cl), picolinic acid (PA), 1-methylimidazole-2-sulfonyl chloride (MLDS), pyridine-3-sulfonyl chloride (PS), (3-dimethylene) aminophenyl) dihydroxyborane (DAPB), 2-bromopyridine-5-boronic acid (BPBA), 2-hydrazinepyridine and hydroxylamine. Another approach is to use quaternary ammonium derivatives that introduce permanently charged groups into the molecule to reduce ionization suppression in ESI sources, such as Girard P and Girard T reagents, quaternary ammonium oxyamine (QAO) reagents and N-methylpyridine-3-sulfonyl chloride (NMPS) reagent.

Compared with Girard reagent, QAO reagent can also derivatize carbonyl group, but its detection sensitivity is higher. Compared with hydroxylamine, the derivatized product of QAO reagent has better stability and higher sensitivity. Therefore, QAO reagents are widely used in the derivatization of carbonyl-containing steroids, but there is no report on their synthesis.

SUMMARY OF THE INVENTION

The invention solves the technical problems of poor stability and low sensitivity of the derivatized product of the carbonyl steroid-containing derivatization reagent in the prior art. The content of the present invention is to provide a synthetic method for derivatizing a carbonyl-containing steroid compound, which is used for derivatizing a carbonyl-containing steroid compound. The synthesis method is to first use 1,3-dibromopropane and N-hydroxyphthalimide to react to synthesize 2-(3-bromopropoxy)isoindoline-1,3-dione; and then use 2 -(3-Bromopropoxy)isoindolin-1,3-dione to prepare 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N, N-trimethylpropylammonium bromide; finally 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropyl bromide ammonium chloride for hydrazinolysis. The synthesis method of the invention has the advantages of low cost, simple steps and high yield, and can derivatize the carbonyl-containing steroid compound, introduce a permanently charged group -N ⁺ Me ₃ into it, and improve the ESI-MS/MS/MS ratio of the carbonyl-containing steroid compound. Detection sensitivity in MS.

According to a first aspect of the present invention, there is provided a method for synthesizing a derivatizing reagent for a carbonyl-containing steroid compound, comprising the following steps:

(1) 2-(3-bromopropoxy)isoindoline-1,3-dione is synthesized by the reaction of 1,3-dibromopropane and N-hydroxyphthalimide;

(2) Reuse 2-(3-bromopropoxy)isoindoline-1,3-dione described in step (1) to prepare 3-((1,3-dioxo-2-isoindoline) dololinyl)-oxy)-N,N,N-trimethylpropylammonium bromide;

(3) 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethyl described in step (2) in methanol or tetrahydrofuran solution The hydrazinolysis is carried out with propyl propyl ammonium bromide to obtain the oxyamine quaternary ammonium salt compound of the structure represented by formula (I);

Preferably, the method for synthesizing 2-(3-bromopropoxy)isoindoline-1,3-dione in step (1) is: combining 1,3-dibromopropane with N-hydroxyphthalic acid The imide is reacted in N,N-dimethylformamide, dimethylsulfoxide or acetonitrile to prepare 2-(3-bromopropoxy)isoindoline-1,3-dione;

In step (2), 2-(3-bromopropoxy) isoindoline-1,3-dione is used to prepare 3-((1,3-dioxo-2-isoindolinyl)-oxygen base)-N,N,N-trimethylpropylammonium bromide method is: react 2-(3-bromopropoxy)isoindoline-1,3-dione with trimethylamine to obtain 3 -((1,3-Dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropylammonium bromide;

In step (3), 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropyl bromide in methanol or tetrahydrofuran solution The method for hydrazinolysis of ammonium is: 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropylammonium bromide in methanol Or react with hydrazine hydrate in tetrahydrofuran solution to obtain the oxyamine quaternary ammonium salt compound of the structure represented by formula (I).

Preferably, the specific method for reacting 1,3-dibromopropane with N-hydroxyphthalimide is: mixing N-hydroxyphthalimide, 1,3-dibromopropane and triethylamine Dissolve in N,N-dimethylformamide, dimethylsulfoxide or acetonitrile solvent, and stir at 20-30℃ for 2-20h; in which N-hydroxyphthalimide and 1,3-di The substance amount ratio of bromopropane is 1:(1-2), and the substance amount ratio of N-hydroxyphthalimide and triethylamine is 1:(1-2).

Preferably, 3-((1,3-dioxo-2-isoindolinyl)-oxy) is prepared using 2-(3-bromopropoxy)isoindolin-1,3-dione The specific method of -N,N,N-trimethylpropylammonium bromide is: dissolving 2-(3-bromopropoxy)isoindoline-1,3-dione in trimethylamine tetrahydrofuran solution, Stir at 35-50°C for 10-20h; the ratio of 2-(3-bromopropoxy)isoindoline-1,3-dione to trimethylamine is 1:(1-3) .

Preferably, 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropylammonium bromide is mixed with hydrazine hydrate in methanol solution The specific method of the reaction is: dissolving 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropylammonium bromide and hydrazine hydrate In methanol or tetrahydrofuran, stirring for 1-5h; wherein 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropyl bromide The mass ratio of ammonium to hydrazine hydrate is 1:(1-5).

In general, compared with the prior art, the above technical solutions conceived by the present invention mainly have the following technical advantages:

(1) The chemical reagents used in the synthesis method are of low cost, the synthesis steps are only three steps, and the yield of the derivatized reagent is greater than 80%, which is conducive to the efficient and rapid production of the derivatized reagent.

(2) The derivatization reagent of the present invention can effectively derivatize carbonyl-containing steroids, introduce permanently charged groups -N ⁺ Me ₃ into them, and improve the detection of carbonyl-containing steroids in ESI-MS/MS sensitivity.

Description of drawings

Fig. 1 is the ¹ HNMR spectrum (400 MHz, solvent is deuterium water D ₂ O) of the purified derivatizing reagent prepared according to the method of Example 1.

FIG. 2 and FIG. 3 are respectively the first-order mass spectrogram and the second-order mass spectrogram of the product after derivatizing 19-norandrosterone by the derivatizing reagent prepared according to the method of Example 1.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

The instruments used for the characterization of the compounds of the present invention are: the mass spectrometer is a Shimadzu LCMS-2020 mass spectrometer, the chromatograph is a Shimadzu high performance liquid chromatograph, and the nuclear magnetic resonance instrument is a Bruker 400MHz nuclear magnetic resonance instrument. The instrument used for the identification of derivatized products is: Thermo Fisher Scientific Q ExactiveTM. Carbonyl-containing steroid standards were purchased from Steraloids. Unless otherwise specified, the chemical reagents used were purchased from Sigma-Aldrich.

The reaction formula of the synthetic carbonyl-containing steroid compound derivatization reagent provided by the invention is as follows:

Example 1

The synthetic method of the carbonyl-containing steroid compound derivatizing reagent of the present invention. In a 50 mL eggplant flask were added N-hydroxyphthalimide (2.01 g, 12.3 mmol), DMF (30 mL), 1,3-dibromopropane (2.5 mL, 24.5 mmol) and triethylamine (3.3 mL, 23.7 mmol), stirred at 25 °C for 16 h. After diluting with water, it was extracted with ethyl acetate. The organic phase was dried, filtered, and subjected to column chromatography (petroleum ether/ethyl acetate = 5/1) to obtain a white solid 2-(3-bromopropoxy)isoindoline- 1,3-Dione 2.3 g, yield 66%. 2-(3-Bromopropoxy)isoindoline-1,3-dione (2.3 g, 8.10 mmol) and trimethylaminetetrahydrofuran solution (24 mL, 24 mmol) were added to a 48 mL sealed tube and stirred at 40°C for 12 h , trimethylamine is dissolved in tetrahydrofuran THF. Filter, collect the solid, wash with dichloromethane to give a white solid 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropyl Ammonium bromide 2.36 g, yield 85%. In a 100mL eggplant flask, add 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropylammonium bromide ((2.36g , 6.88mmol), MeOH (10mL), hydrazine hydrate (8.09g, 13.76mmol), stirred at room temperature for 2h. Filtration, the filtrate was concentrated and further recrystallized with ethanol to obtain a white powder solid 3-(aminooxy)-N, N,N-trimethylpropylammonium bromide 1.30 g, the yield is 80%, and the molecular weight is 213.1190. Figure 1 is the ¹ HNMR spectrum of the purified derivatization reagent prepared according to the method of Example 1 (400 MHz, The solvent is heavy water (D ₂ O). As can be seen from Figure 1, the present invention has successfully prepared quaternary ammonium oxyamine (QAO) compounds.

Example 2

The synthetic method of the carbonyl-containing steroid compound derivatizing reagent of the present invention. In a 50 mL eggplant flask was added N-hydroxyphthalimide (2.01 g, 12.3 mmol), DMSO (30 mL), 1,3-dibromopropane (2.5 mL, 24.5 mmol) and triethylamine (3.3 mL, 23.7 mmol), stirred at 20 °C for 16 h. After diluting with water, it was extracted with ethyl acetate. The organic phase was dried, filtered, and subjected to column chromatography (petroleum ether/ethyl acetate = 5/1) to obtain a white solid 2-(3-bromopropoxy)isoindoline- 1,3-Dione 2.3 g, yield 66%. 2-(3-Bromopropoxy)isoindoline-1,3-dione (2.3 g, 8.10 mmol) and trimethylaminetetrahydrofuran solution (24 mL, 24 mmol) were added to a 48 mL sealed tube and stirred at 35°C for 10 h . Filter, collect the solid, wash with dichloromethane to give a white solid 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropyl Ammonium bromide 2.36 g, yield 85%. In a 100mL eggplant flask, add 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropylammonium bromide ((2.36g , 6.88mmol), THF (10mL), hydrazine hydrate (4.05g, 6.88mmol), stirred at room temperature for 1h. Filtration, the filtrate was concentrated and further recrystallized with ethanol to obtain a white powder solid 3-(aminooxy)-N, N,N-trimethylpropylammonium bromide 1.30 g, the yield is 80%, and the molecular weight is 213.1190.

Example 3

The synthetic method of the carbonyl-containing steroid compound derivatizing reagent of the present invention. In a 50 mL eggplant flask were added N-hydroxyphthalimide (2.01 g, 12.3 mmol), DMF (30 mL), 1,3-dibromopropane (2.5 mL, 24.5 mmol) and triethylamine (3.3 mL, 23.7 mmol), stirred at 30 °C for 16 h. After diluting with water, it was extracted with ethyl acetate. The organic phase was dried, filtered, and subjected to column chromatography (petroleum ether/ethyl acetate = 5/1) to obtain a white solid 2-(3-bromopropoxy)isoindoline- 1,3-Dione 2.3 g, yield 66%. 2-(3-Bromopropoxy)isoindoline-1,3-dione (2.3 g, 8.10 mmol) and trimethylaminetetrahydrofuran solution (24 mL, 24 mmol) were added to a 48 mL sealed tube, and the mixture was stirred at 50°C for 20 h . Filter, collect the solid, wash with dichloromethane to give a white solid 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropyl Ammonium bromide 2.36 g, yield 85%. In a 100mL eggplant flask, add 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropylammonium bromide ((2.36g , 6.88mmol), MeOH (10mL), hydrazine hydrate (20.2g, 34.4mmol), stirred at room temperature for 5h. Filtration, the filtrate was concentrated and further recrystallized with ethanol to obtain a white powder solid 3-(aminooxy)-N, N,N-trimethylpropylammonium bromide 1.30 g, the yield is 80%, and the molecular weight is 213.1190.

Example 4

3-(Aminooxy)-N,N,N-trimethylpropylammonium bromide (16.98mg), methanol (1613μL), and acetic acid (85μL) were added to a 3ml clear glass bottle and vortexed at room temperature Homogeneous and prepared as a solution of 10.00 mg/mL. 19-norandrosterone (1.00 mg) and DMSO (1.00 mL) were added to a 3 ml transparent glass bottle, sonicated and vortexed until all dissolved, to prepare a stock solution with a concentration of 1.00 mg/mL. Acetonitrile (2450 μL) 19-norandrosterone stock solution (1.00 mg/mL, 50 μL) was added to a 5 mL transparent glass bottle to obtain a solution with a 19-norandrosterone concentration of 20.00 ug/mL. A 19-norandrosterone solution (20.00ug/mL, 50μL) was added in acetonitrile (2450μL) to a 5mL transparent glass bottle to obtain a working solution with a concentration of 40.00ng/mL of 19-norandrosterone. Precisely pipet 100μL of 9-norandrosterone solution into a 1.5mL centrifuge tube, add 19-norandrosterone working solution (40.00ng/mL, 100μL), blow dry with nitrogen, add 3-(aminooxy)-N,N , N-trimethylpropylammonium bromide solution (10.00mg/mL, 50μL), vortex and mix, react at room temperature for 1h, add 100μL of 70% methanol, vortex and mix, and centrifuge at high speed for 5min (17000g) , take the supernatant into a 1.5mL centrifuge tube for LC-MS analysis (the injection volume is 10μL).

Analysis was performed using a Shimadzu UPLC/Q-Exactive-MS system (Thermo Fisher). An ultimateXB-C18 column column (100 mm×2.1 mm, 1.8 μm) was used. Mobile phase A was water (containing 0.1% formic acid and 10.0 mM ammonium formate); mobile phase B was acetonitrile. The elution gradient was set as follows: 0-0.01min, 15%B; 0.01-28min, 15%-55%B; 28-38min, 55%-65%B; 38-38.1min, 65%-90%B; 38.1-42 min, 90% B; 42-42.1 min, 90%-15% B; 42.1-45 min, 15% B. The flow rate was set at 0.3 mL/min; the column oven was kept at 40°C. An aliquot of 10 μL of sample was injected into the UPLC/Q-Exactive-MS system. The MS operating parameters were set as follows: positive ion; source temperature 350 °C; applied power supply voltage 3.5 kV; sheath gas flow rate 30 arp, auxiliary gas flow rate 15 arp; capillary temperature 300 °C. The m/z for MS scans was 200–800 with a resolution of 100,000, and the m/z for MS/MS scans was in the 50-M+50 range with a resolution of 500,00.

Raw LC-MS data was acquired in Full Scan-ddMS ² positive ion mode and analyzed using Xcalibur ^™ 4.0 software (Thermo Fisher). The structural formula of the hormone standard 19-norandrosterone before and after derivatization is shown in the figure below. The mass spectrograms of the first stage are shown in Figures 2 and 3, indicating that the synthesized derivatization reagents can successfully derivatize the hormones and detect them by mass spectrometry.

Those skilled in the art can easily understand that the above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, etc., All should be included within the protection scope of the present invention.

Claims (5)

1. a synthetic method for containing carbonyl steroid derivatization reagent, is characterized in that, comprises the following steps: (1) 2-(3-bromopropoxy)isoindoline-1,3-dione is synthesized by the reaction of 1,3-dibromopropane and N-hydroxyphthalimide; (2) Reuse 2-(3-bromopropoxy)isoindoline-1,3-dione described in step (1) to prepare 3-((1,3-dioxo-2-isoindoline) dololinyl)-oxy)-N,N,N-trimethylpropylammonium bromide; (3) 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethyl described in step (2) in methanol or tetrahydrofuran solution The hydrazinolysis is carried out with propyl propyl ammonium bromide to obtain the oxyamine quaternary ammonium salt compound of the structure represented by formula (I);

2. synthetic method as claimed in claim 1 is characterized in that, in step (1), the method for synthesizing 2-(3-bromopropoxy) isoindoline-1,3-dione is: by 1, 3-Dibromopropane reacts with N-hydroxyphthalimide in N,N-dimethylformamide, dimethylsulfoxide or acetonitrile to prepare 2-(3-bromopropoxy)iso indoline-1,3-dione; In step (2), 2-(3-bromopropoxy) isoindoline-1,3-dione is used to prepare 3-((1,3-dioxo-2-isoindolinyl)-oxygen base)-N,N,N-trimethylpropylammonium bromide method is: react 2-(3-bromopropoxy)isoindoline-1,3-dione with trimethylamine to obtain 3 -((1,3-Dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropylammonium bromide; In step (3), 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropyl bromide in methanol or tetrahydrofuran solution The method for hydrazinolysis of ammonium is: 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N-trimethylpropylammonium bromide in methanol Or react with hydrazine hydrate in tetrahydrofuran solution to obtain the oxyamine quaternary ammonium salt compound of the structure represented by formula (I). 3. synthetic method as claimed in claim 2 is characterized in that, the concrete method of 1,3-dibromopropane and N-hydroxyphthalimide reaction is: by N-hydroxyphthalimide Amine, 1,3-dibromopropane and triethylamine were dissolved in N,N-dimethylformamide, dimethylsulfoxide or acetonitrile solvent, and stirred at 20-30℃ for 2-20h; wherein N-hydroxyl The ratio of the amount of phthalimide to 1,3-dibromopropane is 1:(1-2), the ratio of the amount of N-hydroxyphthalimide to triethylamine is 1:(1-2). 4. synthetic method as claimed in claim 2 is characterized in that, utilizes 2-(3-bromopropoxy) isoindoline-1,3-dione to prepare 3-((1,3-dioxo The specific method of -2-isoindolinyl)-oxy)-N,N,N-trimethylpropylammonium bromide is: 2-(3-bromopropoxy)isoindoline-1 ,3-dione was dissolved in trimethylamine tetrahydrofuran solution, stirred at 35-50 ℃ for 10-20h; wherein 2-(3-bromopropoxy)isoindoline-1,3-dione and trimethylamine were mixed The ratio of the amount of substances is 1:(1-3). 5. synthetic method as claimed in claim 2 is characterized in that, 3-((1,3-dioxo-2-isoindolinyl)-oxyl)-N,N,N-trimethyl The specific method of reacting propylpropylammonium bromide with hydrazine hydrate in methanol solution is: 3-((1,3-dioxo-2-isoindolinyl)-oxy)-N,N,N -Trimethylpropylammonium bromide and hydrazine hydrate were dissolved in methanol or tetrahydrofuran, stirred for 1-5h; wherein 3-((1,3-dioxo-2-isoindolinyl)-oxy)- The ratio of the amount of N,N,N-trimethylpropylammonium bromide to hydrazine hydrate is 1:(1-5).

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